Method of inhibiting glycolipid synthesis

ABSTRACT

A method is disclosed for inhibiting the biosynthesis of glycolipids in cells capable of producing glycolipids comprising subjecting said cells to a glycolipid inhibitory effective amount of an N-alkyl derivative of 1,5-dideoxy-1,5-imino-D-glucitol in which said alkyl contains from 2-8 carbon atoms.

This is a division of application Ser. No. 08/321,718, filed Oct. 12,1994, which is a continuation of application Ser. No. 08/061,645, filedMay 13, 1993 now U.S. Pat. No. 5,399,567.

BACKGROUND OF THE INVENTION

This invention relates to a novel method of inhibiting glycolipidsynthesis and, more particular, to the use of N-alkyl derivatives of1,5-dideoxy-l,5-imino-D-glucitol for inhibiting glycolipid biosynthesisin cells capable of producing glycolipids, in which said alkyl groupscontain from about 2-8 carbon atoms.

1,5-Dideoxy-1,5-imino-D-glucitol (which is also known as1-deoxynojirimycin or DNJ) and its N-alkyl derivatives are knowninhibitors of the N-linked oligosaccharide processing enzymes,α-glucosidase I and II. Saunier et al., J. Biol. Chem. 257, 14155-14161(1982); Elbein, Ann. Rev. Biochem. 56, 497-534 (1987). As glucoseanalogs they also have potential to inhibit glucosyltransferases.Newbrun et al., Arch. oral Biol. 28, 516-536 (1983); Wang et al.,Tetrahedron Lett. 34, 403-406 (1993). Their inhibitory activity againstthe glucosidases has led to the development of these compounds asantihyperglycemic agents and antiviral agents. See, e.g., PCT Int'l.Appln. WO 87/03903 and U.S. Pat. Nos.: 4,065,562; 4,182,767; 4,533,668;4,639,436; 4,849,430; 5,011,829;and 5,030,638.

BRIEF DESCRIPTION OF THE INVENTION

In accordance with the present invention, a method is provided forinhibiting the biosynthesis of glycolipids in cells capable of producingglycolipids which comprises treatment of said cells with a glycolipidinhibitory effective amount of an N-alkyl derivative of1,5-dideoxy-1,5-imino-D-glucitol (DNJ) in which said alkyl contains from2-8 carbon atoms and preferably from 4-6 carbon atoms. The length of theN-alkyl chain has been found to be important to said inhibitory activitysince the non-alkylated DNJ and the N-methyl derivative of DNJ were eachfound to be inactive for such inhibition. Thus, a minimum alkyl chainlength of 2 carbon atoms has been found to be necessary for efficacy.

Illustratively, the N-butyl DNJ was also unexpectedly found to be asubstantially more potent inhibitor of glycolipid biosynthesis than itis as an α-glucosidase I inhibitor. That is, it was inhibitory ofglycolipid biosynthesis at relatively low concentrations (about 50 μM)compared to the mM level of concentration in cell culture systems forα-glucosidase I inhibition [Karlsson et al., J. Biol. Chem. 268, 570-576(1993)]. Also illustratively, the N-butyl and N-hexyl derivatives of DNJinhibited the biosynthesis of all glucoceramide basedglycosphingolipids.

The inhibitory effect of these compounds on the biosynthesis ofglycolipids is illustrated herein in myeloid cell lines (e.g., HL-60 andK-562) as well as in lymphoid cell lines (e.g., MOLT-4 and H9). Theseare well-known, widely distributed and readily available human celllines. For example, HL-60 cells are promyelocytic cells described byCollins et al., Nature 270, 347-349 (1977). They are also readilyavailable from the American Type Culture Collection, Rockville, Marylandunder accession number ATCC CCL 240. K-562 cells are of myeloid origindescribed by Lozzio and Lozzio, Blood 45, 321-324 (1975). They are alsoreadily available from the same depository under accession number ATCCCCL 243. MOLT-4 cells are lymphoid cells described in J. Nat'l. CancerInst. 49, 891-895 (1972). They are also readily available from the samedepository under accession number ATCC CRL 1582. H9 cells are oflymphoid origin described by Gallo and Popovic, Science 224, 497-500(1984 ). They are also readily available from the same depository underaccession number ATCC HTB 176.

The inhibition of glycolipid biosynthesis by these N-alkyl derivativesof DNJ is further demonstrated herein by the reduction of the binding ofcholera toxin to these four illustrative cell lines when cultured in thepresence on N-butyl DNJ. These compounds thus are also useful asanti-microbial agents by inhibiting the surface expression on glycolipidreceptors for bacteria and bacterial toxins as illustrated hereinafterin Tables 1 and 2, respectively.

The inhibitory effect upon the biosynthesis of glycolipids is stillfurther illustrated by the ability of N-butyl DNJ to offsetglucoceramide accumulation in a standard, state-of-the-art in vitromodel of Gaucher's disease in which the murine macrophage cell lineWEHI-3B was cultured in the presence of an irreversibleglucocerebrosidase inhibitor, conduritol β epoxide (CBE), to mimic theinherited disorder found in Gaucher's disease. The compound preventslysosomal glycolipid storage which is useful for the management of thisand other glycolipid storage disorders as illustrated hereinafter inTable 3.

Illustratively, the N-butyl-DNJ is also shown herein to be a moreeffective inhibitor of glycolipid biosynthesis than either PDMP or PPMP.PDMP, which chemically isDL-threo-l-phenyl-2-decanoylamino-3-morpholino-1-propanol, is known tobe an effective inhibitor of the glycosyltransferase that makesglucosylceramide. See, for example, Shukla et al., Biochim. Biophys.Acta 1083, 101-108 (1991), and Shukla and Radin, J. Lipid Res. 32,713-722 (1991), for reports on this activity of PDMP. Its analog PPMP,chemically isDL-threo-1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol. Thus, theglycolipid biosynthesis inhibitory activity of N-butyl-DNJ iscorrelatable to the corresponding activity of conventional compounds inthis field.

                  TABLE 1                                                         ______________________________________                                        GLYCOSPHINGOLDIPID RECEPTORS                                                  FOR BACTERIAL CELLS                                                           Microorganism Target Issue                                                                             Presumed Specificity                                 ______________________________________                                        E. coli       Urinary    Galα4Galβ                                 E. coli       Urinary    GlcNAcβ                                         Propionibacterium                                                                           Skin/Intestine                                                                           Galβ4Glcβ                                  Several bacteria                                                                            Diverse    Galβ4Glcβ                                  Streptococcus pneumoniae                                                                    Respiratory                                                                              GlcNAcβ3Gal                                     E. coli CFA/I Intestine  NeuAcα8                                        E. coli       Urinary    NeuAcα3Gal                                     E. coli       Intestine  NeuGcα3Galβ4GlcβCer                                           GalNAcβ4(NeuAcα3)-                                                 Galβ4GlcβCer                               Staphylococcus                                                                              Urinary    Galβ4GlcNAc                                     saprophyticus                                                                 Actinomyces naeslundi                                                                       Mouth      Galβ, GalNAcβ,                                                      Galβ3GalNAcβ,                                                       GalNacβ3Galβ                               Pseudomonas   Respiratory                                                                              GalNAcβ4Gal                                     Neisseria gonorrhoeae                                                                       Genital    Galβ4Glcβ                                                           NeuAcα3Galβ4GlcNAc                        ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                        GLYCOSPHINGOLIPID RECEPTORS                                                   FOR BACTERIAL TOXINS                                                                                          Presumed                                      Micro-                          Receptor                                      organism Toxin      Target Tissue                                                                             Sequence                                      ______________________________________                                        Vibrio   Cholera toxin                                                                            Small Intestine                                                                           Galβ3GalNAcβ4-                      chloerae                        (NeuAcα3)Gal-                                                           β4GlcβCer                           E. coli  Heat-labile                                                                              Intestine   GalβGalNAcβ4-                                toxin                  (NeuAcα3)Gal-                                                           β4GlcβCer                           Clostridium                                                                            Tetanus toxin                                                                            Nerve       Galβ3GalNAcβ4-                      tetani                          (NeuAcα8Neu-                                                            Acα3)Galβ4Glc-                                                     βCer                                     Clostridium                                                                            Botulinum  Nerve       NeuAcα8NeuAcα-                    botulinum                                                                              toxin A and E                                                                            Membrane    3Galβ3GalNAcβ-                                                      4(NeuAcα8Neu-                                                           Acα3)Galβ4Glc-                                                     βCer                                     Clostridium                                                                            Botulinum  Nerve       NeuAcα3Galβ3-                      botulinum                                                                              toxin B, C Membrane    GalNAβ4(Neu-                                      and F                  Acα8NeuAcα3)-                                                     Galβ4GlcβCer                        Clostridium                                                                            Botulinum  Nerve       GalβCer                                  botulinum                                                                              toxin B    Membrane                                                  Clostridium                                                                            Delta toxin                                                                              Cell lytic  GalNAcβ4-                                perfringens                     (NeuAcα3)Galβ-                                                     4GlcβCer                                 Clostridium                                                                            Toxin A    Large       Galα3GalβGlc-                      difficile           Intestine   NAcβ3Galβ4-                                                         GlcβCer                                  Shigella Shiga toxin                                                                              Large       Galα4GalβCer                       dysenteriae         Intestine   Galα4Galβ4Glc-                                                     βCer                                                                     GlcNAcβ4Glc-                                                             NAc                                           E. coli  Vero toxin or                                                                            Intestine   Galα4Galβ4-                                 Shiga-like             GlcβCer                                           toxin                                                                ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                        HERIDITARY GLYCOLIPID STORAGE DISORDERS                                       Disease   Lipid Accumulation                                                                           Enzyme Defect                                        ______________________________________                                        Gaucher's Glucocerebroside                                                                             Glucocerebroside-β-                                                      glucosidase                                          Ceramide  Ceramide Lactoside                                                                           Ceramidelactoside-β-                            Lactoside                galactosidase                                        Lipidosis                                                                     Fabry's   Ceramide Trihexoside                                                                         Ceramidetrihexoside-α-                                                  galactosidase                                        Tay-Sach's                                                                              Ganglioside GM2                                                                              Hexosaminidase A                                     Sandhoff's                                                                              Globoside and GM2                                                                            Hexosaminidase A and B                               General   Ganglioside GM1                                                                              β-Galactosidase                                 Gangliosidosis                                                                Fucosidosis                                                                             H-isoantigen   α-Fucosidase                                   Krabbe's  Galactocerebroside                                                                           Galactocerebroside-β-                                                    galactosidase                                        Metrachromatic                                                                          Sulfatide      Sulfatidase                                          Leukodystrophy                                                                ______________________________________                                    

DETAILED DESCRIPTION OF THE INVENTION AND BRIEF DESCRIPTION OF THEDRAWINGS

While the specification concludes with claims particularly pointing outand distinctly claiming the subject matter regarded as forming theinvention, it is believed that the invention will be better understoodfrom the following illustrative detailed description taken inconjunction with the accompanying drawings in which:

FIG. 1, in two parts A and B, shows by autoradiographic visualizationthe effects on glycolipid biosynthesis in HL-60 cells that weremetabolically labelled with [¹⁴ C] palmitic acid, FIG. 1A: in thepresence of 0.5 mM N-butyl deoxynojirimycin (+NB-DNJ) or FIG. 1B:absence of NB-DNJ (untreated -UT).

FIG. 2 is a bar chart which shows the cholera toxin binding sites percell for four different cell lines (HL-60, H9, K-562 and MOLT-4) inwhich the cholera toxin was fluorescein conjugated and the levels ofbinding to the cell surfaces of untreated (UT) cells and cells treatedwith 0.5 mM N-butyl deoxynojirimycin (+NB-DNJ) were measured by flowcytometry.

FIG. 3 shows by thin layer chromatography (TLC)the effects on WEHI-3Bcells cultured in the presence (+) or absence (-) of an irreversibleglucocerebrosidase inhibitor, conduritol β epoxide (CBE), to mimic theinherited disorder found in Gaucher's disease in which the cells werecultured in the presence (5 to 500 μM) or absence (-) of N-butyldeoxynojirimycin (NB-DNJ) and in which the glucosylceramide (Glc-Cer)levels were examined by TLC analysis.

FIG. 4, in four parts, A, B, C and D, shows the effects as in FIG. 3 butin which the glucosylceramide (Glc-Cer) levels were measured bytransmission electron microscopy instead of TLC. FIG. 4A shows untreatedrepresentative lysosome; FIG. 4B shows lysosome treated with CBE; FIG.4C shows lysosome treated with CBE plus 500 μM plus NB-DNJ; FIG. 4Dshows lysosome treated with CBE plus 50 μM NB-DNJ. The scale bar of FIG.4 is shown in FIG. 4D and represents 0.1 μm.

FIG. 5 is a graphical representation which shows the inhibition ofcholera toxin binding in HL-60 cells cultured in the presence of variousN-alkyl-DNJ compounds as indicated at a range of doses (0.0 to 1.0mg/ml) or untreated (UT) for three days at each dose and assayed by flowcytometry following staining with FITC-cholera cholera toxin. The dataare plotted as dose of compound (mg/ml) on the x-axis against meanchannel fluorescence intensity (mean channel number) on the y-axis.

FIG. 6, in four parts, A, B, C and D, shows by autoradiographicvisualization the effects as in FIG. 1, compared to similar treatmentwith PDMP or PPMP. FIG. 6A: untreated (UT); FIG. 6B: in the presence of0.5 mM N-butyl deoxynojirimycin (NB-DNJ); FIG. 6C: in the presence of 5μM PDMP; FIG. 6D: in the presence of 5 μM PPMP.

In order to further illustrate the invention, the following detailedexamples were carried out although it will be understood that theinvention is not limited to these specific examples or the detailsdescribed therein.

EXAMPLE 1

To investigate the effects of the imino sugar N-butyldeoxynojirimycin(NB-DNJ) on glycolipid biosynthesis, HL-60 cells were metabolicallylabelled with [¹⁴ C]-palmitic acid in the presence or absence of 0.5 mMNB-DNJ. Total cellular lipids were solvent extracted and separated bytwo dimensional thin layer chromatography (2D-TLC) and the individualcomponents visualized by autoradiography (FIG. 1). The major cellularphospholipid species were unaffected by NB-DNJ treatment as verified byTLC spot elution, recovery and scintillation counting. However, both theneutral glycolipids and gangliosides were absent from treated cultures(FIG. 1B). This implied that a very early step in glycolipidbiosynthesis was affected by NB-DNJ treatment. To establish whether thisactivity was a common property of imino sugars and related compounds, anumber of N-linked oligosaccharide processing inhibitors were assayedfor their ability to inhibit HL-60 glycolipid biosynthesis using 2D-TLCanalysis (Table 4). The glucosidase inhibitors DNJ and castanospermine,and the mannosidase inhibitors swainsonine and deoxymannojirimycin(DMJ), had no effect. When the N-alkylated derivatives of DNJ weretested the N-methyl analogue had no effect but both the N-butyl andN-hexyl analogues surprisingly inhibited the biosynthesis ofglycolipids. This indicated that the length of the alkyl chain was acritical parameter for this inhibitory activity. In addition, NB-DNJ wasinhibitory at relatively low compound concentrations (approximately 50μM) indicating that this compound is a more potent inhibitor ofglycolipid biosynthesis than it is as an α-glucosidase I inhibitor (mMrange in cell culture systems). It is believed that the N-butyl andN-hexyl derivatives are specifically inhibitingUDP-glucose:N-acylsphingosine glucosyltransferase [Basu et al., J. Biol.Chem. 248, 1388-1394 (1973)] (EC 2.4.1.80). This transferase is pivotalin generating glucosyl ceramide (Glc-Cer) which is the precursor for themore complex glycosphingolipids and gangliosides. The inhibition of theglucosyltransferase is consistent with the uniform loss of allglycolipid species observed in the presence of the two compounds (FIG.1). In cell free assays NB-DNJ but not DNJ inhibited the transfer ofglucose from UDP-glucose to a ceramide acceptor.

EXAMPLE 2

This example illustrates that glycolipid expression at the cell surfaceis also inhibited in cells cultured in the presence of NB-DNJ. Four celllines (of both myeloid and lymphoid origin) were grown in mediumcontaining 0.5 mM NB-DNJ for three days and the level of cell surfaceGM1 (Galβ3GalNAcβ4(NeuAcα3)-Galβ4Glcβ3Cer) glycolipid expression wasmeasured by flow cytometry. As a specific probe, advantage was taken ofthe GM1 binding specificity of the cholera toxin B chain [van Heyningen,Nature 249, 415-417 (1974); Karlsson, Ann. Rev. Biochem. 58, 309-350(1989)]. The toxin was fluorescein conjugated and the levels of bindingto the cell surface of treated and untreated cell lines was measured(FIG. 2). The number of cholera toxin binding sites per cell wasdetermined by including fluoresceinated microbead standards in theassay. The four cell lines showed different levels of cholera toxinbinding. The two myeloid cell lines (HL-60 and K-562) both expressedapproximately 1×10⁵ copies of cholera toxin binding sites per cell whilethe two lymphoid cell lines (MOLT-4 and H9) expressed approximately2.5-5.0×10⁵ copies per cell. The binding of cholera toxin to the fourcell lines cultured in the presence of NB-DNJ was reduced byapproximately 90% in all cases. This was consistent with the loss of GM1from the cell surface and provided further evidence for the inhibitionof glycolipid biosynthesis by NB-DNJ. It also suggests that imino sugarderivatives have use as potential anti-microbial agents by inhibitingthe surface expression of glycolipid receptors for bacteria andbacterial toxins as shown in Tables 1 and 2, respectively.

EXAMPLE 3

The identification of NB-DNJ and N-hexyl DNJ as novel inhibitors ofglycolipid biosynthesis offers an alternative approach for manipulatingcellular glycolipid levels. The glycolipid storage disorder, Gaucher'sdisease, results from the autosomal inheritance of a defectiveglucocerebrosidase enzyme (β-D-glucosyl-N-acylsphingosineglucohydrolase, EC 3.2.1.45) which prevents the complete catabolism ofGlc-Cer in the lysosome [Barranger and Ginns, The Metabolic Basis ofInherited Disease, 1677-1698 (McGraw-Hill, New York, 1989); Tybulewiczet al., Nature 357, 407-410 (1992); Beutler, Science 256, 794-799(1992)]. However, in contrast with the impaired degradation of Glc-Cer,the rate of glycolipid biosynthesis in these individuals remains normal.As a consequence, Glc-Cer is accumulated over time leading to lysosomalstorage in cells of the monocyte-macrophage system which is diagnosticof this disorder [Parkin and Brunning, Prog. Clin. Biol. Res. 95,151-175 (1982)]. One approach for the management of this and relateddisorders [Neufeld, Ann. Rev, Biochem. 60, 257-280 (1991)] is to usespecific inhibitors of glycolipid biosynthesis [Vunnam and Radin, Chem.Phys. Lipids 26, 265-278 (1980); Inokuchi and Radin, J. Lip. Res. 28,565-571 (1987); Abe et al., J. Biochem. 111 191-196 (1992)] to reducecellular glycolipid production to a level which can be completelycatabolized by the defective glucocerebrosidase, thereby preventingglycolipid accumulation. This example illustrates that glycolipidstorage can be prevented by NB-DNJ in an in vitro model of Gaucher'sdisease. The murine macrophage cell line WEHI-3B was cultured in thepresence of an irreversible glucocerebrosidase inhibitor, conduritol βepoxide (CBE), to mimic the inherited disorder found in Gaucher'sdisease [Newburg et al., Exp. Molec. Pathol. 48, 317-323 (1988)].WEHI-3B cells are described in Cancer. Res. 37, 546-550 (1977), and arereadily available from the American Type Culture Collection, Rockville,Md, under accession number ATCC TIB 68. The WEHI-3B cells were culturedin the presence or absence of NB-DNJ and glucosylceramide levels wereexamined by TLC analysis (FIG. 3). Following CBE treatment the cellsaccumulated Glc-Cer relative to untreated controls. However, in culturescontaining 500 μM or 50 μM NB-DNJ, this accumulation was prevented. Atthe lower dose (50 μM) cultures contained Glc-Cer levels comparable tountreated controls whereas at the highest dose (500 μM) culturescontained almost undetectable levels of Glc-Cer. Cells treated with 5 μMNB-DNJ were identical to CBE treated cells demonstrating that in this invitro Gaucher's disease model a compound dose of 50 μM NB-DNJ willprevent Glc-Cer accumulation. The lysosomes of CBE treated cultures andCBE plus NB-DNJ cultures were examined by transmission electronmicroscopy (FIG. 4). There was evidence of lipid accumulation in thelysosomes of CBE treated cells, FIG. 4B, relative to untreated controls,FIG. 4A, but not in CBE+NB-DNJ treated cultures FIG. 4C, 500 μM and FIG.4D 50 μM, thereby confirming that NB-DNJ prevented CBE inducedglycolipid accumulation by the partial inhibition of glycolipidbiosynthesis.

The identification herein of N-alkyl derivatives of DNJ capable ofmodulating cellular glycolipid levels is useful for the management ofseveral glycolipid storage disorders. These compounds affect Glc-Cerbiosynthesis which is the precursor of glycolipids accumulating in manystorage disorders, independent of the individual enzyme defects of thesediseases (Neufeld supra). See Table 3, hereinbefore, which listshereditary glycolipid storage disorders and their corresponding lipidaccumulation and enzyme defect. In addition, these compounds havetherapeutic use for the treatment of infectious disease agents whichutilize cellular glycolipid receptors (Karlsson, supra) and asmodulators of cell proliferation [Hakomori, Ann. Rev. Biochem. 50,733-764 (1981); Felding-Habermann et al., Biochemistry 29, 6314-6322(1990)], tumor growth [Inokuchi et al., Cancer Lett. 38, 23-30 (1987)]and metastasis [Inokuchi et al., Cancer Res, 50, 6731-6737 (1990);Mannori et al., Int. J. Cancer 45, 984-988 (1990)], where roles forglycolipids have been implicated.

The detailed procedures for obtaining the results of Examples 1 to 3above, as shown by FIGS. 1 to 6 and Table 4 are as follows:

FIG. 1

Effects of NB-DNJ on total HL-60 lipid composition. Lipid identitieswere determined by comparison to authentic lipid standards, differentialchemical detection of phospholipids and glycolipids and laserdesorptionmass spectrometry analysis of the mono and dihexaside species. Lipidswere assigned as follows (untreated cells, FIG. 1A--left hand panel): 1.gangliosides; 2. lysophospatidylcholine; 3. ceramide phosphorylcholine;4. ceramide phosphorylethanolamine; 5. phospatidylcholine; 6.phosphatidylinositol; 7. phosphatidylethanolamine; 8.phosphatidylglycerol; 9. diglycosylceramide; 10. monoglycosylceramine;11. cholesterol/fatty acids/neutral lipids; N and N, are unknowns and 0is the sample origin. Following NB-DNJ treatment (FIG. 1B--right handpanel) species 1 (gangliosides), 9 (diglycosylceramide), 10(monoglycosylceramide) and N* (unknown) were absent. Method: HL-60 cellswere cultured (10 ml) by conventional procedures as previously described[Platt et al., Eur. J. Biochem. 208, 187-193 (1992)] at a seedingdensity of 5×10⁴ cells per ml in the presence or absence of 0.5 mMNB-DNJ (G. D. Searle & Co., Skokie, Ill.) for 24 hours. For labellingand 2D-TLC, the conventional, published method of Butters and Hughes wasfollowed [In vitro 17, 831-838 (1981)]. Briefly, [¹⁴ C]-palmitic acid(ICN-Flow, High Wycombe, Bucks. UK., 56.8 mCi/mmol) was added as asonicated preparation in fetal calf serum (0.5 μCi per ml) and the cellswere cultured for a further three days maintaining NB-DNJ in thecultures. The cells were harvested, washed three times with PBS andextracted in 1 ml chloroform:methanol (2:1 v/v) overnight at 4° C. Theextracts were centrifuged, the chloroform:methanol extract was retainedand the pellet was re-extracted as above for two hours at roomtemperature. Pooled extracts were dried under nitrogen and redissolvedin 50 μl chloroform:methanol (2:1, v/v). One percent of the samplevolume was taken for the determination of radioactivity by scintillationcounting and a 1×10⁶ cpm loaded as a single spot onto a 20 cm² TLC plate(Merck, BDH, Poole, Dorset, U.K.). The samples were separated in thefirst dimension in chloroform:methanol:water (65:25:4) and the platedried overnight under vacuum. Separation in the second dimension wasachieved using a solvent oftetrahydrofuran:dimethoxymethane:methanol:water (10:6:4:1). Plates wereair dried and exposed to Hyperfilm-MP high performance autoradiographyfilm (Amersham International, Amersham, UK).

Table 4

Effects of sugar analogues on HL-60 glycolipid biosynthesis. The dataare summarized from 2D-TLC analysis on each compound at the indicatedconcentrations (see FIG. 1 method, above). Compounds: The synthesis ofalkylated derivatives of DNJ is well known. See, e.g., Fleet et al.,FEBS Lett. 237, 128-132 (1988). DMJ was purchased from BoehringerMannheim (Lewes, Sussex, U.K.), swainsonine and castanospermine wereobtained from Sigma (Poole, Dorset, UK). Compound doses were selectedthat were close to the tolerated upper limit of the individual compoundswhich maintained ninety percent cell viability. HL-60 cells werecultured as described in FIG. 1 procedure, above.

                  TABLE 4                                                         ______________________________________                                        Compound    Dose (mg/ml) Glycolipid Inhibition                                ______________________________________                                        DNJ         0.2          -                                                    N-methyl DNJ                                                                              0.1          -                                                    N-butyl DNJ 0.001        +/-                                                   "          0.01         +                                                     "          0.1          +                                                    N-hexyl DNJ 0.2          +                                                    DMJ         0.06         -                                                    Castanospermine                                                                           0.1          -                                                    Swainsonine 0.1          -                                                    ______________________________________                                    

FIG. 2

Quantitative analysis of cholera toxin binding to human cell linesfollowing three days treatment with NB-DNJ.

Methods: Cells were maintained in logarithmic phase growth in RPMI-1640medium. Cholera toxin B chain (Sigma) was conjugated to fluoresceinisothyocyanate (Sigma) and flow cytometric analysis was carried out byconventional procedure as described by Platt et al., Supra. Analysis wasperformed on a FACScan Cytometer (Becton Dickinson, Sunnyvale Calif.,USA), Data on viable cells were collected on a four decade log₁₀ scaleof increasing fluorescence intensity. The data are presented as meancopy number of cholera toxin bindings sites per cell on the y-axisagainst the four cell line on the x-axis, in the presence or absence of0.5 mM NB-DNJ. The specificity of cholera toxin:cell surface binding wasestablished by inhibiting this interaction with a one hundred fold molarexcess of GM1 derived oligosaccharide, GalβGalNAcβ4(NeuAcβ3)Galβ4Glcβ3Cer. Seventy to ninety percent inhibition wasachieved depending on the individual cell line. A controloligosaccharide (lacto-N-tetarose) was not inhibitory.

FIGS. 3 and 4

Effects of NB-DNJ on an in vitro model of Gaucher's disease. FIG. 3: 1dimensional TLC analysis on WEHI-3B cells treated as indicated. FIG. 4:transmission electron microscopy of WEHI-3B Gaucher cell lysosomes: A.untreated representative lysosome, B. lysosome showing extensiveaccumulation of dense material in the presence of CBE consistent withGlc-Cer accumulation, C. CBE plus 500 μM NB-DNJ and D. CBE plus 50 μMNB-DNJ, each of C and D showing lysosomes with normal density contents.No changes were observed in the lysosomes of cells treated with NB-DNJalone.

Methods: The murine macrophage cell line WEHI-3B was maintained inlogarithmic phase growth for 14 days in RPMI-1640 in the presence orabsence of 50 μM conduritol βepoxide (CBE, Toronto Research Chemicals,Downsview, Canada) with or without NB-DNJ at the indicatedconcentrations. Cells were passaged every three days in the presence ofthe stated concentrations of compounds. Equal cell numbers (5×10⁶) wereharvested, extracted as described hereinbefore (FIG. 1 procedure),separated by one dimensional TLC (first dimension solvent, FIG. 1procedure) and visualized using β-naphthol (1% w/v in methanol) followedby 50% (v/v) sulphuric acid. Similar data were obtained using theindependent mouse macrophage cell line P388D-1. These cells aredescribed in J. Immunol. 114, 894-897 (1975), and are readily availablefrom the American Type Culture collection, Rockville, Md., underaccession number ATCC TIB 63. The authentic Glc-Cer standard from humanGaucher spleen (arrows) was purchased from Sigma.

Cells for electron microscopy were harvested (1×10⁷ cells pertreatment), washed three times in serum free RPMI-1640 medium and fixedin medium containing 2% glutaraldehyde (v/v) on ice for two hours. Cellswere washed in 0.1M cacodylate buffer containing 20 mM calcium chloride(w/v). Fixed cells were stained with 1% osmium tetroxide in 25 mMcacodylate buffer (w/v) containing 1.5% potassium ferrocyanide (w/v) for2 hours on ice. Samples were dehydrated through an ethanol series (50,70, 95 and 100% v/v), transferred to propylene oxide and embedded inEmbed 800 (Electron Microscopy Sciences, PA, USA). The samples werepolymerized at 60° C., sections cut, stained with uranyl acetate/leadcitrate and observed with a Hitachi 600 microscope at 75 v.

FIG. 5

Dose response curves of cholera toxin binding to HL-60 cells followingthree days treatment with various N-alkyl-DNJ compounds. The test methodemployed for FIG. 5 was the same as for FIG. 2, above, but the data areplotted as dose of compound on the x-axis against mean channelfluorescence intensity on the y-axis. The N-methyl, N-ethyl, N-propyl,N-butyl and N-hexyl derivatives of DNJ wre thus tested and compared withthe untreated (UT) control sample.

FIG. 6

Effects of NB-DNJ, PDMP and PPMP on total HL-60 lipid composition. Thetest method employed for FIG. 6 was the same as for FIG. 1, above, butwas extended to include for comparison treatment withDL-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) orDL-threo-1-phenyl-2-hexadecanoylamino-3-morpholino-1propanol (PPMP),both obtained from Matreya Inc., Pleasant Gap, Pa. FIG. 6A: untreatedcells as in FIG. 1A--left panel; FIG. 6B: cells treated with 0.5 mMNB-DNJ as in FIG. 1B--right panel; FIG. 6C: cells treated with 5 μM PDMPfrom 10 mM stock solution in ethanol; FIG. 6D: cells treated with 5 μMPPMP from 10 mM stock solution in ethanol.

In addition to their use as antimicrobial agents and as inhibitors ofglycolipid biosynthesis in cells, the inhibitory agents described hereinalso can be used for administration to patients afflicted withglycolipid storage defects by conventional means, preferably informulations with pharmaceutically acceptable diluents and carriers.These agents can be used in the free amine form or in their salt form.Pharmaceutically acceptable salt derivatives are illustrated, forexample, by the HCl salt. The amount of the active agent to beadministered must be an effective amount, that is, an amount which ismedically beneficial but does not present toxic effects which overweighthe advantages which accompany its use. It would be expected that theadult human daily dosage would normally range from about one to about100 milligrams of the active compound. The preferable route ofadministration is orally in the form of capsules, tablets, syrups,elixirs and the like, although parenteral administration also can beused. Suitable formulations of the active compound in pharmaceuticallyacceptable diluents and carriers in therapeutic dosage form can beprepared by reference to general texts in the field such as, forexample, Remington's Pharmaceutical Sciences, Ed. Arthur Osol, 16th ed.,1980,Mack Publishing Co., Easton, Pa.

Various other examples will be apparent to the person skilled in the artafter reading the present disclosure without departing from the spiritand scope of the invention. It is intended that all such other examplesbe included within the scope of the appended claims.

What is claimed:
 1. The method of inhibiting the biosynthesis ofglycolipids in human cells capable of producing glycolipids comprisingsubjecting said cells in vitro to a glycolipid inhibitory effectiveamount of an N-alkyl derivative of 1,5-dideoxy-1,5-imino-D-glucitol inwhich said alkyl contains from 2-8 carbon atoms.
 2. The method of claim1 in which the alkyl group contains from 4-6 carbon atoms.
 3. The methodof claim 2 in which the alkyl group is butyl.
 4. The method of claim 2in which the alkyl group is hexyl.
 5. The method of claim 1 in which theinhibitory effective amount is from about 50 μM to about 500 μM.
 6. Themethod of claim 1 in which the glycolipid is a glucoceramide basedglycosphingolipid.
 7. The method of claim 1 in which the glycolipid is alysosomal glycolipid.